Method for stabilizing extracellular vesicles

ABSTRACT

The present invention provides stabilization of an extracellular vesicle. Specifically, the present invention provides, for example, a method of stabilizing the extracellular vesicle including mixing an extracellular vesicle-containing sample with a saccharide and a chelating agent.

TECHNICAL FIELD

The present invention relates to a method of stabilizing extracellularvesicle(s), and the like.

BACKGROUND ART

An extracellular vesicle is a microscopic vesicle secreted from varioustypes of cells and having a membrane structure, and exists in bodyfluids such as blood. The extracellular vesicles secretedextracellularly include exosomes, ectosomes, and apoptotic blebs. Sincethe extracellular vesicle contains various substances that play afunction such as intercellular signaling, it has been analyzed for thepurposes of diagnosis, drug discovery and the like. Thus, it is requiredto develop a method of treating the extracellular vesicles useful forsuch analyses. For example, Patent Literature 1 describes: (1)capability of improving a yield of extracellular vesicles in thepresence of EDTA (chelating agent) at a low concentration ofapproximately 3.0 mg/mL (approximately 10 μM) or less and incapabilityof improving the yield of extracellular vesicles in the presence of EDTAat a higher concentration (Example 1 and FIGS. 1A to 1C); (2) preservingextracellular vesicles in the presence of EDTA at low concentration ofapproximately 2.25 mg/ml (approximately 7.7 μM) at a certain temperatureand time (Example 2, FIGS. 2A and 2B, 3A to 3F); and (3) freezing andthawing extracellular vesicles in the presence of EDTA at a lowconcentration of approximately 2.25 mg/ml (approximately 7.7 μM)(Example 3, FIGS. 4A and 4B).

PRIOR ART REFERENCES Patent Literatures

Patent Literature 1: US Patent Application Publication No. 2015/0125864

SUMMARY OF INVENTION Problem to be Solved by the Invention

If extracellular vesicles can be stabilized, such extracellular vesiclesare promising for improvement in preservability thereof and applicationto diagnosis, drug discovery and the like. Therefore, it is an object ofthe present invention to develop a method for stabilizing extracellularvesicle(s).

Solution to Problem

As a result of an extensive study, the inventors of the presentinvention have found that extracellular vesicle(s) in the extracellularvesicle-containing sample can be stabilized by mixing the extracellularvesicle-containing sample with a certain component such as a saccharide,and completed the present invention.

That is, the present invention is as follows.

[1] A method of stabilizing an extracellular vesicle, the methodcomprising mixing an extracellular vesicle-containing sample with asaccharide and a chelating agent.[2] The method according to [1], wherein the extracellular vesicle is anexosome.[3] The method according to [1] or [2], wherein the extracellularvesicle-containing sample is a body fluid or a culture supernatant.[4] The method according to any of [1] to [3], wherein the extracellularvesicle-containing sample is a blood sample.[5] The method according to any of [1] to [4], wherein a concentrationof the saccharide in the mixing is 2.5 to 100 mg/mL.[6] The method according to any of [1] to [5], wherein a concentrationof the chelating agent in the mixing is 1 to 200 mM.[7] A method of stabilizing an extracellular vesicle, the methodcomprising:(1) mixing an extracellular vesicle-containing sample with a saccharide;and(2) freezing a mixture of the extracellular vesicle-containing sampleand the saccharide.[8] The method according to [7], wherein the freezing is freeze-drying.[9] A method of stabilizing an extracellular vesicle, the methodcomprising:(1) mixing an extracellular vesicle-containing sample with a chelatingagent; and(2) freezing a mixture of the extracellular vesicle-containing sampleand the chelating agent.[10] The method according to [9], wherein the freezing is freeze-drying.[11] An extracellular vesicle-stabilizing reagent comprising asaccharide and a chelating agent.[12] An extracellular vesicle-cryopreservation stabilizing reagentcomprising a saccharide or a chelating agent.

Effect of the Invention

The extracellular vesicle(s) can be stabilized in the extracellularvesicle-containing sample by mixing the extracellular vesicle-containingsample with the saccharide. Therefore, the present invention is useful,for example, in preservation of the extracellular vesicle(s).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 includes graphs of yields of exosomes obtained afterfreeze-drying under various buffer conditions in Example 1. Yields ofexosomes obtained without freeze-drying are defined as 100%.

FIG. 2 is a graph of yields of exosomes obtained after freeze-dryingunder various buffer conditions in Example 2. Yields of exosomesobtained without freeze-drying are defined as 100.

FIG. 3A is a graph of particle counts and a particle diameterdistribution measured in a sample (Control) that is obtained beforefreeze-drying a culture supernatant of a human colon carcinoma cell lineSW1116 in Example 3.

FIG. 3B is a graph of particle counts and a particle diameterdistribution measured in a sample (ED/EG) that is obtained beforefreeze-drying the culture supernatant of the human colon carcinoma cellline SW1116 in Example 3.

FIG. 3C is a graph of particle counts and a particle diameterdistribution measured in a sample (Control) that is obtained afterfreeze-drying the culture supernatant of the human colon carcinoma cellline SW1116 in Example 3.

FIG. 3D is a graph of particle counts and a particle diameterdistribution measured in a sample (ED/EG) that is obtained afterfreeze-drying the culture supernatant of the human colon carcinoma cellline SW1116 in Example 3.

FIG. 4A is a diagram depicting results of western blotting with abiotinylated anti-CD9 antibody of samples obtained from animmunoprecipitation method of a serum specimen that is mixed with eachof various chelating agents at different concentrations in ReferenceExample 1.

FIG. 4B is a diagram depicting results of western blotting with thebiotinylated anti-CD9 antibody of samples obtained from theimmunoprecipitation method of a plasma specimen that is mixed with eachof various chelating agents at different concentrations in ReferenceExample 1.

FIG. 5A is a graph (continued on FIG. 5B) of yields of exosomes obtainedafter freeze-drying under various buffer conditions in Example 4. Yieldsof exosomes obtained without freeze-drying are defined as 100%.

FIG. 5B is a graph of yields of exosomes obtained after freeze-dryingunder various buffer conditions in Example 4. Yields of exosomesobtained without freeze-drying are defined as 100%.

FIG. 6 is a graph of yields of exosomes obtained after freeze-dryingunder various buffer conditions in Example 5. Yields of exosomesobtained without freeze-drying are defined as 100%.

FIG. 7 is a graph of yields of exosomes obtained after freeze-drying ahuman serum sample under various buffer conditions in Example 6. Yieldsof exosomes obtained without freeze-drying are defined as 100%.

FIG. 8 is a graph of yields of exosomes obtained after freeze-dryingunder various buffer conditions in Example 7. Yields of exosomesobtained without freeze-drying are defined as 100%.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

1. Method of Stabilizing Extracellular Vesicle(s)

The present invention provides a method of stabilizing an extracellularvesicle, including mixing an extracellular vesicle-containing samplewith a saccharide.

The invention also provides the method of stabilizing the extracellularvesicle, including mixing the extracellular vesicle-containing samplewith a chelating agent.

The invention also provides the method of stabilizing the extracellularvesicle, including mixing the extracellular vesicle-containing samplewith the saccharide and the chelating agent.

The extracellular vesicle is a microscopic vesicle secreted from varioustypes of cells and having a membrane structure. Examples of theextracellular vesicle include exosomes, ectosomes and apoptotic blebs.Preferably, the extracellular vesicle is the exosome. The extracellularvesicle can also be defined by its size. The size of the extracellularvesicle is, for example, 30 to 1000 nm, preferably 50 to 300 nm, andmore preferably 80 to 200 nm. The size of the extracellular vesicle canbe measured by, for example, a method based on Brownian movement of theextracellular vesicle, a light scattering method, and an electricresistance method, and the like. Preferably, the size of theextracellular vesicle is measured by NanoSight LM10 (manufactured byMalvern Instruments). In the case of using the NanoSight LM10, ameasurement time of 30 seconds, three repetition times, and a detectionthreshold of 15 can be employed as measurement conditions. Theextracellular vesicle can also be defined by using an extracellularvesicle marker. Examples of the extracellular vesicle marker includeCD9, carcinoembryonic antigen (CEA), CD81, CD63, heat shock protein(HSP) 70, HSP90, major histocompatibility complex (MHC) I, tumorsusceptibility gene (TSG) 101, lysosome associated membrane protein(LAMP) 1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide,cholesterol, phosphatidylserine, ALIX, Annexins, Caveolin-I,Flotillin-I, Rab protein and EpCAM.

The extracellular vesicle-containing sample is any sample that containsthe extracellular vesicle. Preferably, the extracellularvesicle-containing sample is a biological liquid sample. Theextracellular vesicle-containing sample may be subjected to anothertreatment before being used for the method of the present invention.Examples of such a treatment include centrifugation, extraction,filtration, precipitation, heating, refrigeration, and stirring.

In one embodiment, the extracellular vesicle-containing sample is aculture supernatant. The culture supernatant may be a cell culturesupernatant or a tissue culture supernatant. Examples of the organismfrom which a cell or a tissue to be cultured is derived include animalssuch as mammalian animals (e.g., primates such as humans and monkeys;rodents such as mice, rats and rabbits; farm animals such as cattle,pigs and goats; and working animals such as horses and sheep) and birds(e.g., chickens), insects, microorganisms (e.g., bacteria), plants andfish. Preferably, the organisms are mammalian animals such as humans.

In another embodiment, the extracellular vesicle-containing sample is abody fluid. The body fluid is derived from the organism as describedabove. Examples of the body fluid include blood samples (e.g., wholeblood, serum and plasma), lymph fluid, tissue fluid, cerebrospinalfluid, ascites, saliva, pancreatic liquid, bile, sweat, seminal fluid,urine, tear fluid, mucosal fluid, breast fluid, thoracic fluid,bronchoalveolar lavage fluid and amnion fluid. Preferably, the bodyfluid is the blood.

In another embodiment, the extracellular vesicle-containing sample is amilk or a fruit juice.

The saccharide is a monosaccharide (e.g., aldose and ketose) or apolysaccharide in which two or more monosaccharides are linked by aglycosidic bond. In the present invention, the saccharides also includederivatives and saccharide alcohols. The derivative of the sacchariderefers to a compound in which a hydrogen atom, a hydroxy group or acarbonyl group is substituted with a substituent in the saccharide. Thesaccharide alcohol refers to a compound in which the carbonyl group isreduced in the saccharide.

More specifically, examples of the monosaccharide include trioses (e.g.,glyceraldehyde, dihydroxyacetone), tetroses (e.g., erythrose, threose,erythrose), pentoses (e.g., xylose, ribose, arabinose, lyxose, ribulose,xylulose, apiose), hexoses (e.g., glucose, fructose, galactose, mannose,allose, altrose, gulose, idose, talose, psicose, sorbose, tagatose), andheptoses (e.g., sedoheptulose, coriose). Examples of the aldose includexylose, glucose, galactose, mannose, glyceraldehyde, erythrose, threose,ribose, arabinose, lyxose, allose, altrose, gulose, idose, and talose.Examples of the ketose include fructose, dihydroxyacetone, erythrulose,xylulose, ribulose, psicose, sorbose, tagatose, sedoheptulose andcoriose.

Examples of the polysaccharide include the polysaccharide in which twoor more of the monosaccharides as described above are linked together.Preferably, examples of the polysaccharide include polysaccharides inwhich two or more monosaccharide units in one or plural kinds of arelinked by a glycosidic bond (e.g., one or more glucose and one or morefructose are linked by a glycosidic bond), and the monosaccharide unitsare selected from the group consisting of glucose, fructose, galactose,mannose and xylose. Such a polysaccharide may be a linear polysaccharideor a cyclic polysaccharide. The linear polysaccharide may be a linearoligosaccharide or a linear polymer polysaccharide. In the case that thepolysaccharide is the linear oligosaccharide, the total number ofmonosaccharide units in the polysaccharide may be, for example, 2 to 20,preferably 2 to 10, more preferably 2 to 6, and still more preferably 2to 4. For example, in the case that the polysaccharide is apolysaccharide in which one or more glucose and one or more fructose arelinked by a glycosidic bond, each of the numbers of glucose and fructosein the polysaccharide may be, for example, 1 to 10, preferably 1 to 5,more preferably 1 to 3, and still more preferably 1 or 2. The linearoligosaccharide may be a disaccharide, for example. Examples of thedisaccharide include sucrose, lactose, and trehalose. Particularlypreferably, the disaccharide is sucrose in which one glucose and onefructose are linked by a glycosidic bond. Examples of the linear polymerpolysaccharide include cellulose, amylose, amylopectin, glucomannan,pullulan, galactomannan, inulin, glycogen, chitin, chitosan,glucuronoxylan, arabinoxylan, agarose, carrageenan, pectin, pectinicacid, alginic acid, fucoidan, chondroitin sulfate and hyaluronan. In thecase that the polysaccharide is the cyclic polysaccharide, the totalnumber of monosaccharide units in the polysaccharide may be, forexample, 5 to 100, preferably 5 to 20, more preferably 5 to 10, andstill more preferably 6 to 8. Examples of the cyclic polysaccharideinclude cyclodextrins (α-cyclodextrin, β-cyclodextrin andγ-cyclodextrin).

Examples of the substituent in the saccharide derivative include ahydrogen atom, a hydroxy group, a C₁₋₆ alkyl group, a C₁₋₆ alkenylgroup, a C₁₋₆ alkynyl group, a C₁₋₆ alkyloxy (alkoxy) group, a C₆₋₁₄aromatic hydrocarbon group, a C₁₋₆ alkyl-carbonyl (acyl) group, acarboxy group, a nitro group, an amino group and a cyano group.

The C₁₋₆ alkyl group is an alkyl having one to six carbon atom(s), andmay be linear, branched or cyclic, and the linear or branched alkyl ispreferable. Examples of the C₁₋₆ alkyl group include methyl, ethyl,propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl,2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl. The C₁₋₆ alkylgroup is preferably a C₁₋₄ alkyl group, and more preferably a C₁₋₃ alkylgroup.

The C₁₋₆ alkenyl group is an alkenyl group having one to six carbonatom(s), and may be linear, branched or cyclic, and the linear orbranched alkenyl is preferable. Examples of the C₁₋₆ alkenyl groupinclude ethenyl (vinyl), propenyl, butenyl, pentenyl and hexenyl. TheC₁₋₆ alkenyl group is preferably a C₁₋₄ alkenyl group, and morepreferably a C₁₋₃ alkenyl group.

The C₁₋₆ alkynyl group is an alkynyl group having one to six carbonatom(s), and may be linear, branched or cyclic, and the linear orbranched alkynyl is preferable. Examples of the C₁₋₆ alkynyl groupinclude ethynyl, propynyl, butynyl, pentynyl and hexynyl. The C₁₋₆alkynyl group is preferably a C₁₋₄ alkynyl group, and more preferably aC₁₋₃ alkynyl group.

The C₁₋₆ alkyloxy group is an alkyloxy group having one to six carbonatom(s). Examples of the C₁₋₄ alkyloxy group include methyloxy,ethyloxy, propyloxy, iso-propyloxy, butyloxy, iso-butyloxy,sec-butyloxy, tert-butyloxy, pentyloxy, isopentyloxy, neopentyloxy,1-ethylpropyloxy, hexyloxy, isohexyloxy, 1,1-dimethylbutyloxy,2,2-dimethylbutyloxy, 3,3-dimethylbutyloxy and 2-ethylbutyloxy. The C₁₋₆alkyloxy group is preferably a C₁₋₄ alkyloxy group, and more preferablya C₁ alkyloxy group.

Examples of the C₆₋₁₄ aromatic hydrocarbon group include phenyl,naphthyl, and anthracenyl. The C₆₋₁₄ aromatic hydrocarbon group ispreferably phenyl or naphthyl, and more preferably phenyl.

The C₁₋₆ alkyl-carbonyl (acyl) group is a carbonyl group having the C₁₋₆alkyl group as described above. Examples of the C₁₋₆ alkyl-carbonylgroup include methylcarbonyl (acetyl), ethylcarbonyl, propylcarbonyl,iso-propylcarbonyl, butylcarbonyl, iso-butylcarbonyl, sec-butylcarbonyl,tert-butylcarbonyl, pentylcarbonyl, isopentylcarbonyl,neopentylcarbonyl, 1-ethylpropylcarbonyl, hexylcarbonyl,isohexylcarbonyl, 1,1-dimethylbutylcarbonyl, 2,2-dimethylbutylcarbonyl,3,3-dimethylbutylcarbonyl and 2-ethylbutylcarbonyl. The C₁₋₆alkylcarbonyl group is preferably a C₁₋₄ alkylcarbonyl group, and morepreferably a C₁₋₃ alkylcarbonyl group.

These substituents may be further substituted with other substituents.Examples of the other substituents include hydroxy groups, carboxygroups, nitro groups, amino groups and cyano groups.

Examples of the derivative of saccharide substituted with thesubstituent include cyclic polysaccharides (e.g., cyclodextrin such asα-cyclodextrin, O-cyclodextrin, γ-cyclodextrin and the like), andderivatives of the linear polymer polysaccharide (e.g., cellulose).Examples of the derivative of the cyclic polysaccharide include cyclicpolysaccharides substituted with a substitutable alkyl group (e.g.,methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin). Examples of thederivative of the linear polymer polysaccharide includes a cellulosederivative.

The cellulose derivative is a cellulose derivative in which a hydrogenatom of at least one hydroxy group of the cellulose is substituted witha hydrophilic group. Examples of the hydrophilic group in the cellulosederivative include carboxyalkyl (e.g., carboxy C₁₋₆ alkyl) andhydroxyalkyl (e.g., hydroxy C₁₋₆ alkyl). The hydrophilic group in thecellulose derivative is preferably carboxyalkyl or hydroxyalkyl.

Examples of the carboxyalkyl include carboxymethyl, carboxyethyl(1-carboxyethyl and 2-carboxyethyl), carboxypropyl (1-carboxypropyl,2-carboxypropyl and 3-carboxypropyl), carboxyisopropyl(1-carboxy-2-methylethyl) and 2-carboxy-2-methylethyl), carboxybutyl(1-carboxybutyl, 2-carboxybutyl, 3-carboxybutyl and 4-carboxybutyl),carboxy t-butyl, carboxypentyl (1-carboxypentyl, 2-carboxypentyl,3-carboxypentyl, 4-carboxypentyl and 5-carboxypentyl), carboxyhexyl(1-carboxyhexyl, 2-carboxyhexyl, 3-carboxyhexyl, 4-carboxyhexyl,5-carboxyhexyl and 6-carboxyhexyl).

Examples of the hydroxyalkyl include hydroxymethyl, hydroxyethyl(1-hydroxyethyl and 2-hydroxyethyl), hydroxypropyl (1-hydroxypropyl,2-hydroxypropyl and 3-hydroxypropyl), hydroxyisopropyl(1-hydroxy-2-methylethyl and 2-hydroxy-2-methylethyl), hydroxybutyl(1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl and 4-hydroxybutyl),hydroxy t-butyl, hydroxypentyl (1-hydroxypentyl, 2-hydroxypentyl,3-hydroxypentyl, 4-hydroxypentyl and 5-hydroxypentyl) and hydroxyhexyl(1-hydroxyhexyl, 2-hydroxyhexyl, 3-hydroxyhexyl, 4-hydroxyhexyl,5-hydroxyhexyl and 6-hydroxyhexyl).

Specific examples of the cellulose derivative include carboxymethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose orhydroxypropylmethyl cellulose.

The cellulose derivative also includes a nanocellulose derivative. Thenanocellulose derivative is a derivative of the nanocellulose describedbelow.

The nanocellulose is a fibrous cellulose having a fiber width in ananometer order. The fiber width of the nanocellulose is, for example,500 nm or less, and may be preferably 200 nm or less, more preferably100 nm or less, still more preferably 50 nm or less, still further morepreferably 10 nm or less, and particularly preferably 5 nm or less.

Examples of the cellulose derivatives also include salts thereof.Examples of the salt includes salts of metal (e.g., a monovalent metalsuch as lithium, sodium, potassium, rubidium and cesium; and a bivalentmetal such as calcium, magnesium and zinc), and salts of inorganic base(e.g., ammonia).

Examples of the saccharide alcohol include: the monomer in which thecarbonyl group is reduced in the monosaccharide as described above; amultimer in which the monomers are linked together, for example, byglycosidic bonds; and a multimer in which the monomer and themonosaccharide as constituent units are linked together, for example, byglycosidic bonds. Examples of the monomer include tritols (e.g.,glycerin), tetritols (e.g., erythritol and threitol), pentitols (e.g.,xylitol, arabinitol and ribitol), hexitols (e.g., sorbitol, mannitol,iditol and galactitol), heptitols (e.g., volemitol and perseitol), andoctyltols (e.g., erythrogalactooctytol). Examples of the saccharidealcohol include: monomers such as xylitol, sorbitol, mannitol,galactitol, fucitol, volemitol, arabinitol, glycerin, iditol,erythritol, threitol, ribitol and the like; and dimers such as lactitoland maltitol.

In the present invention, a mixture of two or more (e.g., two, three,four and five) kinds of saccharides may be mixed with the extracellularvesicle-containing sample. The present invention may include adding thesaccharide to the extracellular vesicle-containing sample before mixingthe extracellular vesicle-containing sample with the saccharide. Thesaccharide may be added simultaneously with or separately from thechelating agent.

In the case of mixing the extracellular vesicle-containing sample withthe saccharide, the concentration of the saccharide in the mixing is notparticularly limited as long as the extracellular vesicle(s) can bestabilized. Such a concentration varies depending on factors such askinds of the saccharides, and may be, for example, 2.5 mg/ml or more,preferably 3.0 mg/mL or more, more preferably 3.5 mg/mL or more, stillmore preferably 4.0 mg/mL or more, still further more preferably 4.5mg/mL or more, and particularly preferably 5.0 mg/mL or more, 6.0 mg/mLor more, 8.0 mg/mL or more or 10.0 mg/mL or more. Also, such aconcentration varies depending on factors such as the kinds ofsaccharides, and may be, for example, 600 mg/mL or less, 400 mg/mL orless, 200 mg/mL or less, or 100 mg/mL or less, and may be preferably 95mg/mL or less, more preferably 90 mg/mL or less, still more preferably85 mg/mL or less, still further more preferably 80 mg/mL or less, andparticularly preferably 75 mg/mL or less, 70 mg/mL or less, 60 mg/mL orless, or 50 mg/mL or less. More specifically, the concentration of thesaccharide varies depending on factors such as the kinds of saccharides,and may be, for example, 2.5 to 800 mg/mL, 2.5 to 600 mg/mL, 2.5 to 400mg/mL, 2.5 to 200 mg/mL or 2.5 to 100 mg/mL, and may be preferably 3.0to 95 mg/mL, more preferably 3.5 to 90 mg/mL, still more preferably 4.0to 85 mg/mL, still further more preferably 4.5 to 80 mg/mL, andparticularly preferably 5.0 to 75 mg/mL, 6.0 to 70 mg/mL, 8.0 to 60mg/mL or 10.0 to 50 mg/mL.

The chelating agent is a compound having a coordination moiety capableof coordinating with a metal ion, or a salt thereof. The number of thecoordination moiety(ies) is preferably 2 or more, more preferably 3 ormore (e.g., 3 or 6). Examples of the coordination atom as thecoordination moiety include an oxygen atom, a phosphorus atom, anitrogen atom, a sulfur atom and a chlorine atom. The coordination atomis preferably the oxygen atom or the phosphorus atom, and morepreferably the oxygen atom. Examples of a coordination group as thecoordination moiety include a group having the abovementionedcoordination atom. The coordination group is preferably a carboxylicacid group or a phosphoric acid group, and more preferably thecarboxylic acid group.

Examples of the chelating agent include ethylenediaminetetraacetic acid(EDTA), glycoletherdiaminetetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), hydroxyethyl iminodiacetic acid(HIDA), nitrilotriacetic acid (NTA), oxalic acid,ethylenediaminetetra(methylene phosphonic acid) (EDTMP) and saltsthereof. Examples of the salts include metal salts (e.g., monovalentmetal salts such as sodium salts, potassium salts, and bivalent metalsalts such as calcium salts, magnesium salts), inorganic salts (e.g.,halide salts such as fluoride, chloride, bromide and iodide, andammonium salts), organic salts (e.g., ammonium salts substituted with analkyl group), and acid addition salts (e.g., salts with an inorganicacid such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitricacid, phosphoric acid and the like, and salts with an organic acid suchas acetic acid, oxalic acid, lactic acid, citric acid,trifluoromethanesulfonic acid, trifluoroacetic acid and the like). Inthe present invention, a mixture of 2 or more (e.g., 2, 3, 4, or 5)kinds of chelating agents may be mixed with the extracellularvesicle-containing sample. The present invention may include adding thechelating agent to the extracellular vesicle-containing sample beforemixing the extracellular vesicle-containing sample with the chelatingagent.

In the case of mixing the extracellular vesicle-containing sample withthe chelating agent, a concentration of the chelating agent in themixing is not particularly limited as long as the extracellularvesicle(s) can be stabilized. Such a concentration varies depending onfactors such as kinds of the chelating agents, and may be, for example,1 mM or more, preferably 5 mM or more, more preferably 10 mM or more,still more preferably 15 mM or more, still further more preferably 20 mMor more, and particularly preferably 30 mM or more, 40 mM or more, or 50mM or more. Also, such a concentration varies depending on factors suchas the kinds of chelating agents, and may be, for example, 200 mM orless, preferably 180 mM or less, more preferably 170 mM or less, stillmore preferably 160 mM or less, still further more preferably 150 mM orless, and particularly preferably 140 mM or less, 120 mM or less, or 100mM or less. More specifically, the concentration of the chelating agentmay be, for example, 1 mM to 200 mM, preferably 5 to 180 mM, morepreferably 10 to 170 mM, still more preferably 15 to 160 mM, stillfurther more preferably 20 to 150 mM, and particularly preferably 30 to140 mM, 40 to 120 mM, or 50 to 100 mM.

The extracellular vesicle-containing sample may be mixed with both ofthe saccharide and the chelating agent.

In the case of using both the saccharide and the chelating agent in thepresent invention, concentrations of the saccharide and the chelatingagent to be used in the mixing can also be defined by their ratio. Forexample, the concentration of the chelating agent per 10 mg/mL of thesaccharide in the mixing varies depending on factors such as the kindsof saccharide and chelating agent, and may be, for example, 1 mM ormore, preferably 5 mM or more, preferably 10 mM or more, still morepreferably 15 mM or more, still further more preferably 20 mM or more,and particularly preferably 30 mM or more, 40 mM or more, or 50 mM ormore. Also, such a concentration varies depending on factors such as thekinds of saccharide and chelating agent, and may be, for example, 200 mMor less, preferably 180 mM or less, more preferably 170 mM or less,still more preferably 160 mM or less, still further more preferably 150mM or less, and particularly preferably 140 mM or less, 120 mM or less,or 100 mM or less. More specifically, the concentration of the chelatingagent may be, for example, 1 mM to 200 mM, preferably 5 to 180 mM, morepreferably 10 to 170 mM, still more preferably 15 to 160 mM, stillfurther more preferably 20 to 150 mM, and particularly preferably 30 to140 mM, 40 to 120 mM, or 50 to 100 mM.

The mixing can be carried out in any way. For example, in the case ofusing both the saccharide and the chelating agent in the presentinvention, the mixing can be carried out simultaneously or separately.More specifically, the extracellular vesicle-containing sample may be(1) mixed with the saccharide and the chelating agent simultaneously,(2) mixed with the chelating agent after being mixed with thesaccharide, or (3) mixed with the saccharide after being mixed with thechelating agent. From the viewpoint of convenience in processing and soon, preferably, the extracellular vesicle-containing sample may be mixedwith the saccharide and the chelating agent simultaneously.

A mixing temperature may be, for example, 4 to 37° C., and preferably 15to 30° C. A mixing time is not particularly limited as long as theextracellular vesicle(s) can be stabilized, and can be controlled asappropriate. The extracellular vesicle-containing sample may be left tostand after being mixed with a certain component such as the saccharide.

Whether the extracellular vesicle is stabilized by the mixing with thecertain component such as the saccharide can be assessed, for example,by: comparing an index value that is measured after the mixing theextracellular vesicle with the certain component such as the saccharide,with a control value measured (under the same condition other than thepresence or absence of the certain component such as the saccharide) forthe extracellular vesicle in absence of the certain component such asthe saccharide; and determining whether the index value is superior tothe control value. For example, stability of a freeze-dried product maybe assessed by: freeze-drying with a freeze dryer (manufactured by TokyoRikakikai Co., Ltd.) by setting a program at −28° C. for 2 hours, at−10° C. for 4 hours and at 20° C. subsequently; and comparing the indexvalues of the freeze-dried products in the presence and absence of thecertain component such as the saccharide. For example, as such an indexvalue, it is possible to utilize an amount of an extracellularvesicle-marker and a measurement value of the number of particlescorresponding to the extracellular vesicle.

The amount of the extracellular vesicle-marker can be measured by anywell-known method in the relevant field.

In the case that the extracellular vesicle-marker is a protein, examplesof the measurement method include immunoassay and mass spectrometry.Examples of the immunoassay include a direct competitive method, anindirect competitive method and a sandwich method. Also, examples ofsuch an immunoassay include chemiluminescent immunoassay (CLIA) [e.g., achemiluminescent enzyme immunoassay (CLEIA)], turbidimetric immunoassay(TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA,indirect competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA),latex agglutination reaction method, fluorescence immunoassay (FIA), andimmunochromatography, Western blotting, immunostaining and fluorescenceactivated cell sorting (FACS). In the case of detecting multiplecomponents, proteomic analysis may be performed.

In the case that the extracellular vesicle-marker is a nucleic acid,examples of the measurement method include a hybridization method usinga probe, a gene amplification method using a primer(s) (e.g., 2, 3 or 4primers) and mass spectrometry.

In the case that the extracellular vesicle-marker is a component otherthan the protein and the nucleic acid, examples of the measurementmethod include immunoassays and mass spectrometric method. In the caseof detecting multiple components, metabolome analysis may be performed.

The number of particles corresponding to the extracellular vesicle(s)can be measured, for example, with an instrument such as a particleanalysis instrument, an electron microscope, a flow cytometer and thelike. Preferably, such a measurement of particle counts can be performedby NanoSight LM10 (manufactured by Malvern Instruments Ltd.). In thecase of using NanoSight LM10, it is possible to employ measurementconditions of 30 seconds measurement time, three repeating times and adetection threshold of 15.

The method of the present invention may further include freezing themixture.

That is, the present invention further provides the method ofstabilizing the extracellular vesicle. The method further includes:

(1) mixing the extracellular vesicle-containing sample with thesaccharide; and(2) freezing the mixture of the extracellular vesicle-containing sampleand the saccharide.

The present invention further provides the method of stabilizing theextracellular vesicle. The method further includes:

(1) mixing the extracellular vesicle-containing sample with thechelating agent; and(2) freezing the mixture of the extracellular vesicle-containing sampleand the chelating agent.

The present invention further provides the method of stabilizing theextracellular vesicle. The method further includes:

(1) mixing the extracellular vesicle-containing sample with thesaccharide and the chelating agent; and(2) freezing the mixture of the extracellular vesicle-containing sample,the saccharide and the chelating agent.

The freezing may be either the freeze-drying or a freezing of solution(non-freeze-drying). The freeze-drying is preferred in the case ofpreserving the extracellular vesicle(s) for a longer period.

The examples are described above, as to the kinds of the saccharide andthe chelating agent as well as their concentrations in the mixing.

2. Extracellular Vesicle-Stabilizing Reagent

The present invention also provides an extracellular vesicle-stabilizingreagent. The reagent of the present invention contains the saccharide orthe chelating agent. Preferably, the reagents of the present inventionmay contain the saccharide and the chelating agent. The saccharide andthe chelating agent are the same as those described above. The reagentof the present invention may include another component (e.g., anothercomponent useful in the stabilization of extracellular vesicle(s)), inaddition to the saccharide and chelating agents.

In one embodiment, the reagent of the present invention is anextracellular vesicle-stabilizing composition containing the saccharide.In another embodiment, the reagent of the present invention is anextracellular vesicle-stabilizing composition containing a chelatingagent. In yet another embodiment, the composition of the presentinvention is an extracellular vesicle-stabilizing composition containingboth the saccharide and the chelating agent.

In order to stabilize the extracellular vesicle(s), the reagent of thepresent invention can be mixed with the extracellular vesicle-containingsample for use. For the purpose of preserving the extracellularvesicle(s) stably, the mixture of the extracellular vesicle-containingsample and the reagent of the present invention may be frozen. In thiscase, the reagent of the present invention can be used as anextracellular vesicle-cryopreservation stabilizing reagent. The freezingcan be either the freeze-drying or the freezing of solution(non-freeze-drying). The freeze-drying is preferred in the case ofpreserving the extracellular vesicle(s) for a longer period.

The concentration of the saccharide in the composition is notparticularly limited, as long as the extracellular vesicle(s) can bestabilized after the mixing with the extracellular vesicle-containingsample. Such a concentration varies depending on factors such as thekind of the saccharide, and may be, for example, 2.7 mg/mL or more,preferably 3.0 mg/mL or more, more preferably 3.5 mg/mL or more, stillmore preferably 4.0 mg/mL or more, still further more preferably 4.5mg/mL or more, and particularly preferably 5.0 mg/mL or more, 6.0 mg/mLor more, 8.0 mg/mL or more, or 10.0 mg/mL or more. Also, such aconcentration varies depending on factors such as the kind ofsaccharide, and may be, for example, 600 mg/mL or less, 400 mg/mL orless, 200 mg/mL or less, or 100 mg/mL or less, preferably 95 mg/mL orless, more preferably 90 mg/mL or less, still more preferably 85 mg/mLor less, still further more preferably 80 mg/mL, particularly preferably75 mg/mL or less, 70 mg/mL or less, 60 mg/mL or less, or 50 mg/mL orless. More specifically, the concentration of the saccharide variesdepending on factors such as the kind of saccharide, and may be, forexample, 2.7 to 800 mg/mL, 2.7 to 600 mg/mL, 2.7 to 400 mg/mL, 2.7 to200 mg/mL or 2.7 to 100 mg/mL, preferably 3.0 to 95 mg/mL, morepreferably 3.5 to 90 mg/mL, still more preferably 4.0 to 85 mg/mL, stillfurther more preferably 4.5 to 80 mg/mL, and particularly preferably 5.0to 75 mg/mL, 6.0 to 70 mg/mL, 8.0 to 60 mg/mL or 10.0 to 50 mg/mL.

The concentration of the chelating agent in the composition is notparticularly limited, as long as the extracellular vesicle(s) can bestabilized after the mixing with the extracellular vesicle-containingsample. Such a concentration varies depending on factors such as thekind of the chelating agent, and may be, for example, 1.1 mM or more,preferably 5 mM or more, preferably 10 mM or more, preferably 15 mM ormore, more preferably 20 mM or more, still more preferably 30 mM ormore, still further more preferably 40 mM or more, and particularlypreferably 50 mM or more. Also, such a concentration varies depending onfactors such as the kind of chelating agents, and may be, for example,200 mM or less, preferably 180 mM or less, more preferably 170 mM orless, still more preferably 160 mM or less, still further morepreferably 150 mM or less, and particularly preferably 140 mM or less,120 mM or less, or 100 mM or less. More specifically, the concentrationof the chelating agent may be, for example, 1.1 mM to 200 mM, preferably5 to 180 mM, more preferably 10 to 170 mM, still more preferably 15 to160 mM, still further more preferably 20 to 150 mM, and particularlypreferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.

In the case that the composition of the present invention contains boththe saccharide and the chelating agent, the concentrations of thesaccharide and the chelating agent can also be defined by their ratio.For example, the concentration of the chelating agent per 10 mg/mL ofthe saccharide varies depending on factors such as the kinds ofsaccharide and chelating agent, and may be, for example, 1 mM or more,preferably 5 mM or more, preferably 10 mM or more, preferably 15 mM ormore, more preferably 20 mM or more, still more preferably 30 mM ormore, still further more preferably 40 mM or more, and particularlypreferably 50 mM or more. Also, such a concentration varies depending onfactors such as the kinds of saccharide and chelating agents, and maybe, for example, 200 mM or less, preferably 180 mM or less, morepreferably 170 mM or less, still more preferably 160 mM or less, stillfurther more preferably 150 mM or less, and particularly preferably 140mM or less, 120 mM or less, or 100 mM or less. More specifically, theconcentration of the chelating agent may be, for example, 1 mM to 200mM, preferably 5 to 180 mM, more preferably 10 to 170 mM, still morepreferably 15 to 160 mM, still further more preferably 20 to 150 mM, andparticularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.

The composition of the present invention is dissolved in an aqueoussolution for use. The composition of the present invention may be eithera non-aqueous composition (e.g., mixture powder) containing thesaccharide and/or the chelating agent or an aqueous solution containingthe saccharide and/or the chelating agent. From the viewpoint of quickand simple use and so on, the aqueous solution containing the saccharideand/or the chelating agent is preferred. Examples of the aqueoussolution include water (e.g., distilled water, sterilized water,sterilized distilled water and pure water) and buffer. The buffer ispreferred. Examples of the buffer include phosphate buffer,phosphate-buffered saline (PBS), tartrate buffer, citrate buffer,acetate buffer, glycine buffer, carbonate buffer,2-morpholinoethanesulfonic acid (MES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, borate buffer,3-morpholinoproponesulfonic acid (MOPS) buffer,N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer,N,N-bis(2-hydroxyethyl)glycine (Bis-Tris) buffer and2-[4-(2-hydroxyethyl)1-piperazinylethanesulfonic acid (HEPES) buffer. Itis preferable that the buffer has neutral pH. More specifically, such apH is preferably 5.0 or more, more preferably 5.5 or more, and stillmore preferably 6.0 or more. Also, the pH is preferably 9.0 or less,more preferably 8.5 or less, and still more preferably 8.0 or less. ThepH can be measured by well-known methods in the relevant field.Preferably, it is possible to employ a value measured at 25° C. with apH meter having a glass electrode, as the pH.

The composition of the present invention can be used by being mixed withthe extracellular vesicle-containing sample as appropriate. A mixingratio of the composition of the present invention and the extracellularvesicle-containing sample (the extracellular vesicle-containingsample/the composition) is, for example, 1/30 to 30, preferably 1/20 to20, more preferably 1/10 to 10, and still more preferably 1/10 to 1.

In the case that the composition of the present invention is the aqueoussolution, a volume of the aqueous solution is, for example, 1 μL to 100mL. Preferably, the volume of the aqueous solution may be 10 μL or more,100 μL or more, or 1000 μL or more. Also, the volume of the aqueoussolution may be 50 mL or less, 10 mL or less, or 2 mL or less.

In another embodiment, the reagent of the present invention is a kitincluding the saccharide and/or the chelating agent. The saccharideand/or the chelating agent can be provided in a solid form or in anaqueous solution form, and is preferably provided in the aqueoussolution form. Therefore, the kit of the present invention may beprovided in an aqueous solution form containing the saccharide, may beprovided in an aqueous solution form containing the chelating agent, maybe provided in an aqueous solution form containing both the saccharideand the chelating agent, or may be provided in a first aqueous solutionform containing the saccharide and a second aqueous solution formcontaining the chelating agent. The aqueous solution is described above,and preferably the buffer. Examples and pH of the buffer are describedabove. The abovementioned concentrations of saccharide and chelatingagent in the composition of the present invention can be applied also toa concentration of the saccharide in the first aqueous solution, aconcentration of the chelating agent in the second aqueous solution anda concentration ratio of the saccharide in the first aqueous solutionand the chelating agent in the second aqueous solution. Theabovementioned mixing ratios and the volumes in the composition of thepresent invention can be applied also to a mixing ratio of the first andsecond aqueous solutions and the extracellular vesicle-containing sampleand volumes of the first and second aqueous solutions.

3. Mixture Containing Extracellular Vesicle(s)

The present invention also provides a mixture containing the saccharideand/or the chelating agent, and the extracellular vesicle. The mixtureof the present invention may further include the aqueous solution (e.g.,the buffer solution described above). The form of the mixture is notparticularly limited, and preferably the aqueous solution or a frozenproduct thereof (e.g., a freeze-dried product). The mixture of thepresent invention can be obtained by treating the extracellularvesicle-containing sample in the method of the present invention or withthe reagent thereof as described above. For example, the mixture of thepresent invention is useful in preservation of the extracellularvesicle(s).

The concentrations described above regarding the method of the presentinvention can be applied also to a concentration of the saccharide orthe chelating agent in the mixture, and a concentration ratio of thesaccharide and the chelating agent. The concentration (particlecounts/mL) of extracellular vesicle(s) in the mixture is, for example,1×10² to 1×10¹⁵, preferably 1×10³ to 1×10¹⁴, more preferably 1×10⁴ to1×10¹³, still more preferably 1×10⁵ to 1×10¹², and particularlypreferably 1×10⁶ to 1×10¹¹.

In the case that the mixture of the present invention is the aqueoussolution, volume of the aqueous solution is, for example, 1 μL to 100mL. Preferably, the volume of aqueous solution may be 10 μL or more, 100μL or more, or 1000 μL or more. Also, the volume of aqueous solution maybe 50 mL or less, 10 mL or less, or 2 mL or less.

EXAMPLES

Hereinafter, the present invention will be described with reference toExamples, but the present invention is not limited to these Examples.

Example 1: Stabilization of Exosome with Chelating Agent

The influence of chelating agent on freeze-drying of exosomes wasinvestigated.

1) Recovery of Exosomes

A culture supernatant of human non-small cell lung carcinoma cell lineH1299 cultured in serum-free medium for three days was used as a sample.The culture supernatant was centrifuged at 2,000×g at 4° C. for 5minutes, then filtered through a 0.22 μm filter (manufactured byMillipore Corp.), and then concentrated using Amicon Ultra-15(manufactured by Millipore Corp.). The concentrate was centrifuged at20,000×g at 4° C. for 15 minutes. Then, the supernatant was centrifugedat 100,000×g at 4° C. for 1 hour. The supernatant was discarded, andthen PBS (2.9 mM NaH₂PO₄, 9.0 mM Na₂HPO₄, 137 mM NaCl) or 50 mM EDTA/50mM EGTA/PBS was added to resuspend the precipitate. Then, theresuspension was centrifuged at 100,000×g at 4° C. for 1 hour. Thesupernatant was discarded, and PBS or 50 mM EDTA/50 mM EGTA/PBS(recovery buffer) was newly added to resuspend the precipitate in orderto recover exosomes.

2) Freeze-Drying of Exosomes

Protein quantification was performed for the recovered exosomes withQubit (trademark) Protein Assay Kit (manufactured by Thermo FisherScientific Inc.). Then, the recovery buffer was further added to each ofthe recovered exosomes so as to prepare solutions containing the sameconcentration of protein. Next, 1 volume of the recovered exosomes wasmixed with 24 volumes of the freeze-drying buffer. After dispensation,the solution was freeze-dried with a freeze dryer (manufactured by TokyoRikakikai Co., Ltd.) with a program of −28° C. for 2 hours, −10° C. for4 hours and subsequent 20° C., in order to obtain a freeze-dried productcontaining freeze-dried exosomes. As the freeze-drying buffer, PBS orEDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) was used. pH of PBSused was 7.4 (same in Examples 1 to 3).

Subsequently, the freeze-dried product was dissolved in milli-Q water(manufactured by Millipore Corp.) or 50 mM EDTA/50 mM EGTA/H₂O(dissolving buffer).

3) Assessment of Stability of Exosomes Based on Measurement of Amount ofExosome-Specific Antigen (CD9)

The amount of the dissolved freeze-dried exosomes was measured by ELISAsystem. Specifically, PBS (pH 7.4) containing anti-CD9 antibody preparedin the applicant was added to a 96-well ELISA plate (manufactured byNUNC Inc.), and incubated overnight at 4° C. Then, each well was washedthree times with PBS containing 0.05 wt % of Tween (registeredtrademark) 20 (PBS-T), 200 μL of PBS containing 0.5 wt % Casein wasadded, and incubated for 2 hours at room temperature. After washing withPBS-T, 100 μL of the dissolved freeze-dried product diluted with PBS wasadded to each well and incubated for 1 hour at 37° C. Then, afterwashing with PBS-T, 100 μL of PBS containing biotinylated anti-CD9antibody prepared in the applicant and streptavidin conjugated alkalinephosphatase (SA-ALP, manufactured by GeneTex Co.) was added to eachwell, and then incubated for 1 hour at 37° C. Then, after washing withPBS, 100 μL of Lumipulse (registered trademark) substrate solution(manufactured by Fujirebio Inc.) was added to be allowed to react for 10minutes at 37° C., and then emission count was measured at a 477 nmwavelength. The count of the sample obtained without freeze-drying isdefined as 100%.

As a result, in the freeze-drying buffer containing the chelating agent,exosome amount was increased (FIG. 1).

Therefore, it was demonstrated that the chelating agent can stabilizethe exosomes in the freeze-drying of exosomes.

Example 2: Stabilization of Exosome with Sucrose

The influence of saccharide on the freeze-drying of exosomes wasinvestigated.

1) Recovery of Exosome

A culture supernatant of human colon carcinoma cell line SW480 culturedin the serum-free medium for three days was used as a sample. Theculture supernatant was centrifuged at 2,000×g at 4° C. for 5 minutes,then filtered through the 0.22 μm filter (manufactured by MilliporeCorp.), and then concentrated using Amicon Ultra-15 (manufactured byMillipore Corp.). The concentrate was centrifuged at 20,000×g at 4° C.for 15 minutes. Next, the supernatant was centrifuged at 100,000×g at 4°C. for 1 hour. The supernatant was discarded, and then PBS was added toresuspend the precipitate. Then, the resuspension was centrifuged at100,000×g at 4° C. for 1 hour. The supernatant was discarded, and PBSwas newly added to resuspend the precipitate in order to recoverexosomes.

2) Freeze-Drying of Exosome

Protein quantification was performed for the recovered exosomes withQubit (trademark) Protein Assay Kit (manufactured by Thermo FisherScientific Inc.). Then, PBS was further added to each of the recoveredexosomes to prepare solutions containing the same concentration ofprotein. Next, 1 volume of the recovered exosomes was mixed with 24volumes of the freeze-drying buffer. After dispensation, the solutionwas freeze-dried with the above-mentioned freeze dryer with a program of−28° C. for 2 hours, −10° C. for 4 hours and subsequent 20° C., in orderto obtain a freeze-dried product containing freeze-dried exosomes. Asthe freeze-drying buffer, PBS, EDTA/EGTA/PBS (final conc. 50 mM EDTA/50mM EGTA), sucrose/PBS (final conc. 10 mg/mL sucrose), orEDTA/EGTA/sucrose/PBS (final conc. 50 mM EDTA/50 mM EGTA/10 mg/mLsucrose) was used.

Subsequently, the freeze-dried product was dissolved in milli-Q water(manufactured by Millipore Corp.).

3) Assessment of Stability of Exosome Based on Measurement of Amount ofExosome-Specific Antigen (CD9)

The amount of the dissolved freeze-dried exosomes was measured by ELISAsystem. Specifically, PBS (pH 7.4) containing anti-CD9 antibody preparedin the applicant was added to the 96-well ELISA plate (manufactured byNUNC Inc.), and incubated overnight at 4° C. Then, each well was washedthree times with PBS-T, 200 μL of PBS containing 0.5 wt % Casein wasadded, and incubated for 2 hours at room temperature. After washing withPBS-T, 100 μL of the dissolved freeze-dried product diluted with PBS wasadded to each well and incubated for 1 hour at 37° C. Then, afterwashing with PBS-T, 100 μL of PBS containing biotinylated anti-CD9antibody prepared in the applicant and streptavidin conjugated alkalinephosphatase (SA-ALP, manufactured by GeneTex Co.) was added to eachwell, and then incubated for 1 hour at 37° C. Then, after washing withPBS, 100 μL of Lumipulse substrate solution was added to be allowed toreact for 5 minutes at 37° C., and then emission count was measured at a477 nm wavelength. The count of the sample obtained without thefreeze-drying is defined as 100.

As a result, in the freeze-drying buffer containing the sucrose, theexosome amount was increased (FIG. 2). Also, in the freeze-drying buffercontaining the sucrose and the chelating agent, the exosome amount wasfurther increased.

Therefore, it was demonstrated that the saccharide can stabilize theexosomes in the freeze-drying of exosomes. In addition, it wasdemonstrated that the stability of the exosomes with the saccharide canbe enhanced in the presence of the chelating agent.

Example 3: Assessment of Stability of Freeze-Dried Exosome Based onMeasurement of Particle Counts

The stability was assessed on the basis of particle counts of thefreeze-dried exosomes by measurement using NanoSight for a freeze-driedsample.

1) Recovery of Exosome

A culture supernatant of human colon carcinoma cell line SW1116 culturedin the serum-free medium for three days was used as a sample. Theculture supernatant was centrifuged at 2,000×g at 4° C. for 5 minutes,then filtered through the 0.22 μm filter (manufactured by MilliporeCorp.), and then concentrated using Amicon Ultra-15 (manufactured byMillipore Corp.). The concentrate was centrifuged at 20,000×g at 4° C.for 15 minutes. Next, the supernatant was centrifuged at 100,000×g at 4°C. for 1 hour. The supernatant was discarded, and then PBS (Control) or50 mM EDTA/50 mM EGTA/PBS (ED/EG) was added to resuspend theprecipitate. Then, the resuspension was centrifuged at 100,000×g at 4°C. for one hour. The supernatant was discarded, and PBS or 50 mM EDTA/50mM EGTA/PBS (ED/EG) was newly added to resuspend the precipitate inorder to recover exosomes.

2) Freeze-Drying of Exosome

Protein quantification was performed for the recovered exosomes withQubit (trademark) Protein Assay Kit (manufactured by Thermo FisherScientific Inc.). Then, the recovery buffer was further added to each ofthe recovered exosomes to prepare solutions containing the sameconcentration of protein. Next, 1 volume of the recovered exosomes wasmixed with 24 volumes of the freeze-drying buffer. After dispensation,the solution was freeze-dried with the abovementioned freeze dryer witha program of −28° C. for 2 hours, −10° C. for 4 hours and subsequent 20°C., in order to obtain a freeze-dried product containing freeze-driedexosomes. As the freeze-drying buffer, PBS and EDTA/EGTA/PBS (finalconc. 50 mM EDTA/50 mM EGTA) (ED/EG) was used.

Subsequently, the freeze-dried product was dissolved in milli-Q water(manufactured by Millipore Corp.).

3) Assessment of Stability of Freeze-Dried Exosome

The number and size distribution of the particles were measured byNanoSight LM10 (manufactured by Malvern Instruments) for the dissolvedfreeze-dried exosomes. The measurement was performed for 30 seconds andrepeated three times. The analysis was carried out with a detectionthreshold of 15.

As a result, the number of particles with 100 to 200 nm diameterconsidered to be exosomes was increased in the freeze-drying buffercontaining the chelating agent (Table 1 and FIGS. 3A to 3D).

Therefore, it was demonstrated that the chelating agent can stabilizethe exosomes in the freeze-drying of exosomes also by the measurement ofthe number of particle.

TABLE 1 Number of particles corresponding to exosomes before and afterfreeze-drying Pa/mL Control ED/EG Before freeze-drying 9.32 × 10⁸ 1.66 ×10⁹ After freeze-drying 4.08 × 10⁸ 8.83 × 10⁸ vs. before freeze-drying44% 53%

Reference Example 1: Treatment of Exosomes with Various Chelating Agents

For chelating agents, disodium ethylenediaminetetraacetate (EDTA-2Na),glycoletherdiaminetetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), hydroxyethyliminodiacetic acid(HIDA), nitrilotriacetic acid (NTA), oxalic acid dihydrate,ethylenediaminetetra (methylenephosphonic acid) (EDTMP) (EDTA-2Na, EGTAwere from Dojindo Molecular Technologies, Inc., HEDTA, HIDA, NTA, EDTMPwere from Tokyo Chemical Industry Co., Ltd., and oxalic acid dihydratewas from Wako Pure Chemical Industries, Ltd.) were used. The influencesof each chelating agent on the precipitation amounts of exosomes wereinvestigated.

300 μL of a serum specimen or a plasma specimen was diluted with 600 μLof PBS or each chelating agent-added PBS. The concentrations of eachchelating agent to be used were 1, 10, and 50 mM at final concentrations(provided, 1, 10, and 30 mM only for EDTMP). Then, the solutions wereleft to stand at room temperature for 30 minutes, and then centrifugedat 20,000×g at 4° C. for 15 minutes. The resulting supernatant wastransferred to a new tube, and magnetic beads (Protein G Dynabeads)immobilized with 2 μg of the monoclonal antibody recognizing CD9prepared in the applicant were added thereto. After reacting at 4° C.overnight, the magnetic beads were washed three times with PBS, and thesamples were eluted with the sample buffer (containing SDS) from themagnetic beads to use as samples for western blotting. These sampleswere analyzed by western blotting using the biotinylated anti-CD9antibody.

As a result, all of the chelating agents used showed recovery ofexosomes by immunoprecipitation both in the serum specimens (FIG. 4A)and the plasma specimens (FIG. 4B). In addition, as the concentration ofthe chelating agent increased, the amount of the immunoprecipitatedexosomes increased. In particular, the immunoprecipitation amounts ofexosomes drastically increased under the conditions of 30 mM EDTMP, 50mM EDTA, EGTA, HEDTA, NTA and oxalic acid (FIGS. 4A and 4B).

Example 4: Stabilization of Exosome with Various Saccharides

The influence of various saccharides on the freeze-drying of exosomeswas investigated.

1) Recovery of Exosome

The culture supernatant of the human non-small cell lung carcinoma cellline H1299 cultured in the serum-free medium for three days was used asa sample. The exosomes were recovered by the recovery method in Example2.

2) Freeze-Drying of Exosome

Protein quantification was performed for the recovered exosomes withQubit (trademark) Protein Assay Kit (manufactured by Thermo FisherScientific Inc.). Then, PBS was further added to each of the recoveredexosomes to prepare solutions containing the same concentration ofprotein. Next, 1 volume of the recovered exosomes was mixed with 9volumes of the freeze-drying buffer. After dispensation, the solutionwas freeze-dried with a freeze dryer (manufactured by Genevac Ltd.) inorder to obtain a freeze-dried product containing freeze-dried exosomes.As the freeze-drying buffer, PBS and PBS solutions, each of thatcontains one of lactose, fructose, methyl β-cyclodextrin (MPCD),hydroxypropyl β-cyclodextrin (HpβCD), γ-cyclodextrin (γCD), glucose,lactitol, sorbitol, xylitol, sucrose, trehalose, mannitol and xylose ina final concentration of 2.0 mg/mL, 10 mg/mL and 50 mg/mL, were used.

Subsequently, the freeze-dried product was dissolved in milli-Q water(manufactured by Millipore Corp.).

3) Assessment of Stability of Exosome Based on Measurement of Amount ofExosome-Specific Antigen (CD9)

The amount of the dissolved freeze-dried exosomes was measured by ELISAsystem. Specifically, PBS (pH 7.4) containing anti-CD9 antibody preparedin the applicant was added to the 96-well ELISA plate (manufactured byNUNC Inc.), and incubated overnight at 4° C. Then, each well was washedthree times with PBS-T, 200 μL of PBS containing 0.5 wt % BSA was added,and incubated for 2 hours at room temperature. After washing with PBS-T,100 μL of the dissolved freeze-dried product was added to each well andincubated for 1 hour at 37° C. Then, after washing with PBS-T, 100 μL ofPBS containing biotinylated anti-CD9 antibody prepared in the applicantwas added to each well, and then incubated for 1 hour at 37° C. Then,after washing with PBS-T, 100 μL of PBS containingstreptavidin-conjugated alkaline phosphatase (SA-ALP, manufactured byGeneTex, Inc.) was added to each well, and then incubated for 1 hour at37° C. Then, after washing with PBS-T, 100 μL of Lumipulse substratesolution was added to be allowed to react for 5 minutes at 37° C., andthen emission count was measured at a 480 nm wavelength. The count ofthe sample obtained without the freeze-drying is defined as 100%.

As a result, in the freeze-drying buffers each containing thesaccharide, the exosome amount was increased (FIGS. 5A and 5B). Inparticular, most of the investigated saccharides increased the exosomeamount in the freeze-drying buffers each containing 10 mg/mL or 50 mg/mLof the saccharide.

Therefore, it was demonstrated that each of the saccharides canstabilize the exosomes in the freeze-drying of exosomes, and inparticular, exhibit a high stabilization effect in the case of 10 to 50mg/mL of the saccharide.

Example 5: Stabilization of Exosome in Combination Use of VariousSaccharides and Chelating Agent

The influence of combination use of various saccharides and chelatingagent on the freeze-drying of exosomes was investigated.

1) Exosome Recovery, Freeze-Drying of Exosome and Assessment ofStability of Exosome Based on the Measurement of Amount ofExosome-Specific Antigen (CD9)

The exosome recovery, the freeze-drying of exosome and assessment ofstability of exosomes were carried out by the method described inExample 4 other than the freeze-dying buffer.

PBS, EDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) (ED/EG), oreither one thereof that contains lactose, γ-cyclodextrin (γCD), glucoseor lactitol in a final concentration of 10 mg/mL, was used as thefreeze-drying buffer.

As a result, in the freeze-drying buffers containing the saccharide andthe chelating agent, the exosome amount was increased (FIG. 6).

Therefore, it was demonstrated that the stabilization of exosomes by thesaccharide can be enhanced in the presence of the chelating agent.

Example 6: Stabilization of Exosome Derived from Serum

The influence of various saccharides, the chelating agent or acombination use thereof on the freeze-drying of exosomes derived fromserum was investigated.

1) Recovery of Exosome

Human serum was used as a sample. The serum was centrifuged at 2,000×gat 4° C. for 5 minutes, and then centrifuged at 20,000×g at 4° C. for 15minutes. Next, the supernatant was centrifuged at 100,000×g at 4° C. for3 hours. The supernatant was discarded, and then PBS was added toresuspend the precipitate. Then, the resuspension was centrifuged at100,000×g at 4° C. for 1 hour. The supernatant was discarded, and PBSwas newly added to resuspend the precipitate in order to recoverexosomes.

2) Freeze-Drying of Exosome and Assessment of Stability of Exosome Basedon Measurement of Amount of Exosome-Specific Antigen (CD9)

The freeze-drying of exosomes and assessment of stability of exosomeswere carried out by the method described in Example 4 other than thefreeze-dying buffer.

PBS, EDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) (ED/EG), oreither one thereof that contains lactose, glucose or lactitol in a finalconcentration of 10 mg/mL or 50 mg/mL, was used as the freeze-dryingbuffer.

As a result, in the freeze-drying buffers containing the saccharides orthe chelating agent, the exosome amount was increased (FIG. 7). Theexosome amount was further increased in the case of using thefreeze-drying buffers containing both of the saccharide and thechelating agent.

Therefore, it was demonstrated that the stabilization effect of exosomesby the saccharide and the chelating agent was proven to be enhanced alsofor the exosomes derived from the serum.

Example 7: Stabilization of Exosomes with Carboxymethyl Cellulose

The influence of carboxymethyl cellulose (CMC) on the freeze-drying ofexosomes was investigated.

1) Recovery of Exosome

The culture supernatant of the human non-small cell lung carcinoma cellline H1299 cultured in the serum-free medium for three days was used asa sample. The culture supernatant was centrifuged at 2,000×g at 4° C.for 5 minutes, then filtered through the 0.22 μm filter (manufactured byMillipore Corp.), and then concentrated using Amicon Ultra-15(manufactured by Millipore Corp.). The concentrate was centrifuged at100,000×g at 4° C. for 3 hours. Next, the supernatant was discarded, andthen PBS was added to resuspend the precipitate. Then, the resuspensionwas centrifuged at 100,000×g at 4° C. for 1 hour. The supernatant wasdiscarded, and PBS was newly added in order to recover exosomes.

2) Freeze-Drying of Exosome

Protein quantification was performed for the recovered exosomes withQubit (trademark) Protein Assay Kit (manufactured by Thermo FisherScientific Inc.), and concentration was adjusted. Next, 1 volume of therecovered exosomes was mixed with an equivalent volume of thefreeze-drying buffer, frozen at −20° C. for 3 hours, and then dried witha centrifugal evaporator (manufactured by Scrum Inc.). Then, thefreeze-dried product was dissolved in milli-Q water (manufactured byMillipore Corp.) and diluted with PBS. PBS and CMC/PBS (final conc. 0.2wt % or 1 wt % CMC) were used as the freeze-drying buffers.

3) Assessment of Stability of Exosome Based on Measurement of Amount ofExosome-Specific Antigen (CD9)

The amount of the dissolved freeze-dried exosomes was evaluated by ELISAsystem in which the anti-CD9 antibody prepared in the applicant was usedin a solid phase and the biotinylated anti-CD9 antibody prepared in theapplicant and SA-ALP (manufactured by GeneTex, Inc.) were used fordetection. In the evaluation, the count of the sample obtained withoutthe freeze-drying was defined as 100%.

As a result, under the condition containing 0.2 wt CMC or 1 wt % CMC inthe freeze-drying, residual amount of exosomes is increased (FIG. 8).

Therefore, it was demonstrated that CMC can stabilize the exosomes inthe freeze-drying of exosomes.

1. A method of stabilizing an extracellular vesicle, the methodcomprising mixing an extracellular vesicle-containing sample with asaccharide and a chelating agent.
 2. The method of claim 1, wherein theextracellular vesicle is an exosome.
 3. The method of claim 1, whereinthe extracellular vesicle-containing sample is a body fluid or a culturesupernatant.
 4. The method of claim 1, wherein the extracellularvesicle-containing sample is a blood sample.
 5. The method of claim 1,wherein a concentration of the sacchande in the mixing is 2.5 to 100mg/mL.
 6. The method of claim 1, wherein a concentration of thechelating agent in the mixing is 1 to 200 mM.
 7. A method of stabilizingan extracellular vesicle, the method comprising: (1) mixing anextracellular vesicle-containing sample with a saccharide, to obtain amixture; and (2) freezing the mixture.
 8. The method of claim 7, whereinthe freezing comprises freeze-drying.
 9. A method of stabilizing anextracellular vesicle, the method comprising: (1) mixing anextracellular vesicle-containing sample with a chelating agent, toobtain a mixture; and (2) freezing the mixture.
 10. The method of claim9, wherein the freezing comprises freeze-drying. 11-12. (canceled)
 13. Astabilized extracellular vesicle obtained by the method of claim
 1. 14.A stabilized extracellular vesicle obtained by the method of claim 7.15. A stabilized extracellular vesicle obtained by the method of claim9.